Project Summary: Tuberculosis (TB), caused by Mycobacterium tuberculosis infects about 2 billion- a third- of the world's population. The success of the pathogen can be attributed to its extraordinary ability to survive indefinitely in the host. The treatment of TB requires a prolonged regimen of three anti-tuberculosis drugs to ensure complete eradication of a sub-population that continues to persist against antibiotics. The emergence of multi-drug resistant and extremely drug resistant TB is a tremendous concern as it completely eliminates all treatment options as well as overturns the effort made by WHO to control the spread of this disease. Therefore an understanding of the mechanism of persistence of M. tuberculosis inside the host is imperative for designing a short and effective treatment against the disease. During the course of infection M. tuberculosis is challenged with a variety of host defense mechanisms which the pathogen has to overcome. Of these, the challenges faced during the early phase of infection are perhaps the most critical for the pathogen to survive so as to establish a successful infection. This primarily constitutes the respiratory burst that occurs upon phagocytosis of the bacteria by alveolar macrophages and results in the generation of reactive oxygen and nitrogen intermediates. Whereas most pathogenic bacteria are cleared by these antimicrobial activities in the infected macrophages, M. tuberculosis has evolved mechanisms to subvert these challenges as well as to actively repair the damage caused. The bases in DNA are particularly susceptible to damage by these reactive oxygen and nitrogen species- the bacteria must therefore possess active mechanisms to repair the damage in order to ensure the survival of the pathogen. Previous work suggests that mycobacteria possess most of the DNA damage repair systems utilized by other bacteria; in addition it has a number of novel genes and pathways as well. This is not very surprising considering that M. tuberculosis resides in an environment rich in agents that can damage DNA. The project here proposes to identify novel DNA stress response pathways in the pathogen which will help in better understanding the mechanisms utilized by the bacteria to repair its DNA and are simultaneously very attractive as therapeutic targets. Our preliminary work has identified several novel genes that have previously not been shown to be involved in DNA damage repair in the surrogate host M. smegmatis. While the genes identified from M. smegmatis provide an invaluable insight into mycobacterial survival strategies against oxidative stress and strongly support the hypothesis that key repair pathways remain to be discovered, it is likely that M. tuberculosis could have additional genes for its survival because of its obligate intracellular growth requirement. In this revised submission we propose to identify novel DNA damage repair pathways in the pathogen, M. tuberculosis as well as study the involvement of regulatory proteins identified in the preliminary screen.) ) PUBLIC HEALTH RELEVANCE: Project)Narrative) ) Tuberculosis,)caused)by)M.)tuberculosis)infects)nearly)a)third)of)the)world's) population)and)kills)about)2)million)people)in)the)world)every)year.)Developing)new) drugs)for)the)disease)requires)a)better)understanding)of)the)mechanisms)the) pathogen)utilizes)to)persist)in)the)challenging)environment.)In)this)project)we)study) the)novel)mechanisms)used)by)bacteria)to)counter)the)constant)assault)on)its)DNA) by)the)reactive)oxygen)species)present)in)the)oxidizing)environment)of)macrophages) where)it)resides)for)prolonged)periods)of)time.))